PROJECT SUMMARY There currently exists a fundamental gap in our understanding of the mechanisms that underlie the specification of cardiovascular subtypes, such as atrial and ventricular cardiomyocytes, epicardium, endothelium smooth muscle cells during development. This represents an important problem because it prevents the complete comprehension, and therefore also the treatment of congenital heart disease, which affects ~1% of newborns. Importantly, this knowledge gap hinders the translational approach of generating defined cardiovascular cell types from human pluripotent stem cells (hPSCs) based on the concept of reproducing known principles of normal development in culture. The long-term goal of our work is to better understand the underlying mechanisms directing specification and differentiation of the cardiovascular lineages. The overall objective of this application is to identify the specific mechanisms that determine ventricular cell fate specification and differentiation during mouse development in vivo and to translate these insights to the hPSC differentiation system to efficiently generate human ventricular cardiomyocytes in vitro. The central hypothesis is that ventricular progenitor cells can be identified, isolated and characterized early during development, by lineage-tracing of Foxa2, and that this progenitor population can be reproduced during hPSC differentiations. This hypothesis has been formulated based on preliminary data produced in the applicant's laboratory, demonstrating that prospective ventricular cells can be identified as early as during gastrulation, using Foxa2 as a marker. Foxa2 expression and subsequent lineage-tracing enable monitoring of these cells over the course of their specification and differentiation. In Aim 1, prospective ventricular cells will be isolated and analyzed molecularly at key stages during development to identify the relevant signaling pathways and gene regulatory mechanisms responsible for ventricular specification and differentiation. In Aim 2, the hPSC model system will be used, along with ventricular-specific reporter cell lines, cell surface antibody screening technology, and small molecule pathway modulations to establish robust protocols for the generation and characterization of pure human ventricular cardiomyocytes. This approach effectively combines complementary model systems to examine specification mechanisms of ventricular cells during heart development. We expect results from our studies to advance the understanding of heart development by uncovering key regulators of early cardiac specification. Ultimately, such knowledge has the potential to have a broad translational impact in the understanding of congenital heart disease, and for the generation of therapeutically relevant and safe cell populations from hPSCs.